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Experimental Eye Research 90 (2010) 478e492

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Experimental Eye Research

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Anatomy of the human corneal innervation

Carl F. Marfurt*, Jeremiah Cox 1, Sylvia Deek 1, Lauren DvorscakIndiana University School of Medicine e Northwest, 3400 Broadway, Gary, IN 46408, United States

a r t i c l e i n f o

Article history:Received 4 November 2009Accepted in revised form 16 December 2009Available online 29 December 2009

Keywords:corneal nervessubbasal nerves

* Corresponding author. Tel.: þ1 219 980 6666; faxE-mail addresses: cmarfurt@iun.edu (C.F. Marfurt),

deek@comcast.net (S. Deek), ldvorsca@iun.edu (L. Dv1 These two authors contributed equally to this wo

0014-4835/$ e see front matter � 2009 Elsevier Ltd.doi:10.1016/j.exer.2009.12.010

a b s t r a c t

The anatomy of the human corneal innervation has been the subject of much investigation; however,a comprehensive description remains elusive. The purpose of the present study was to provide a detaileddescription of the human corneal innervation using a novel approach involving immunohistochemicallystained anterior-cornea whole mounts. Sixteen donor corneas aged 19e78 years were cut with a 6.0 mmtrephine into a central plug and two peripheral rims. Each specimen was sectioned serially on a cryostatto produce several 100 mm-thick stromal sections and a 100e140 mm-thick anterior-cornea whole mountthat contained the entire corneal epithelium and much of the anterior stroma. The corneal innervationwas stained with a primary antibody against beta neurotubulin and subjected to rigorous quantitativeand qualitative analyses. The results showed that a mean of 71.3 � 14.3, uniformly spaced, main stromalnerve bundles entered the cornea at the corneoscleral limbus. The bundles averaged 20.3 � 7.0 mm indiameter, were separated by a mean spacing of 0.49 � 0.40 mm, and entered the cornea at a meandistance of 293 � 106 mm from the ocular surface. Each stromal bundle gave rise through repetitivebranching to a moderately dense midstromal plexus and a dense subepithelial plexus (SEP). The SEP wascomprised of modest numbers of straight and curvilinear nerves, most of which penetrated Bowman'smembrane to supply the corneal epithelium, and a more abundant and anatomically complex populationof tortuous, highly anastomotic nerves that remained largely confined in their distribution to the SEP. SEPdensity and anatomical complexity varied considerably among corneas and was less dense and patchierin the central cornea. A mean of 204 � 58.5 stromal nerves penetrated Bowman's membrane to supplythe central 10 mm of corneal epithelium (2.60 nerves/mm2). The density of Bowman's membranepenetrations was greater peripherally than centrally. After entering the epithelium, stromal nervesbranched into groups of up to twenty subbasal nerve fibers known as epithelial leashes. Leashes in thecentral and intermediate cornea anastomosed extensively to form a dense, continuous subbasal nerveplexus, while leashes in the peripheral cornea demonstrated fewer anastomoses and were less complexanatomically. Viewed in its entirety, the subbasal nerve plexus formed a gentle, whorl-like assemblage oflong curvilinear subbasal fibers, 1.0e8.0 mm in length, that converged on an imaginary seam or gentlespiral (vortex) approximately 2.51 � 0.23 mm inferonasal to the corneal apex. Mean subbasal nerve fiberdensity near the corneal apex was 45.94 � 5.20 mm/mm2 and mean subbasal and interconnecting nervefiber diameters in the same region were 1.51 � 0.74 mm and 0.69 � 0.26 mm, respectively. Intraepithelialterminals originated exclusively as branches of subbasal nerves and terminated in all epithelial layers.Nerve terminals in the wing and squamous cell layers were morphologically diverse and ranged in totallength from 9 to 780 mm. The suprabasal layers of the central corneal epithelium contained approxi-mately 605.8 terminals/mm2. The results of this study provide a detailed, comprehensive description ofhuman corneal nerve architecture and density that extends and refines existing accounts. An accurate,detailed model of the normal human corneal innervation may predict or help to understand theconsequences of corneal nerve damage during refractive, cataract and other ocular surgeries.

� 2009 Elsevier Ltd. All rights reserved.

: þ1 219 980 6566.coxjj@iun.edu (J. Cox), sylvia.orscak).rk.

All rights reserved.

1. Introduction

The human cornea is the most densely innervated surface tissuein the body. In addition to their important sensory functions,corneal nerves help maintain the functional integrity of the ocularsurface by releasing trophic substances that promote cornealepithelial homeostasis and by activating brainstem circuits that

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stimulate reflex tear production and blinking. Consequently,damage to corneal nerves as the result of surgery, trauma or diseaseleads to diminished corneal sensitivity and possible transient orlong-term alterations in the functional integrity of the ocularsurface.

The anatomy of the human corneal innervation has been studiedfor many years by a variety of methods, including light and electronmicroscopy, immunohistochemistry and in vivo confocal micros-copy (IVCM). Despite these efforts, a detailed, comprehensivedescription of human corneal nerve architecture remains elusive.Light and electron microscopic investigations of human cornealnerve distribution, density, and ultrastructure (Al-Aqaba et al.,2009; Muller et al., 1996, 1997; Schimmelpfennig, 1982; Ueda et al.,1989; Zander and Weddell, 1951) have generated most of the dataon which current models of corneal innervation are based (Mulleret al., 2003). More recently, IVCM has been used to image theinnervation in healthy and diseased corneas and has providedconsiderable new information on the morphology, density, anddisease- or surgical-induced alterations of corneal nerves, withspecial emphasis on the subbasal nerve plexus (Oliveira-Soto andEfron, 2001; Patel and McGhee, 2005; see Patel and McGhee, 2009,for review and additional references). IVCM is especially useful forimaging corneal nerves near the apex because of the relative ease ofobtaining good quality tangential images in this region; however,although IVCM provides excellent resolution, it is often incapable ofimaging reliably corneal epithelial terminals and very small diam-eter subbasal and stromal nerves.

The purpose of the present study was to provide a detailed andcomprehensive description of the human corneal innervation usinga novel approach that involved immunohistochemical staining of100e140 mm-thick “anterior-cornea whole mounts” and stromalsections. This method provides excellent visualization of the mainstromal bundles, midstromal plexus, subepithelial plexus, subbasalnerve plexus, and intraepithelial terminals throughout the entirecornea. The results of this study were presented in preliminaryfashion at ARVO (Marfurt et al., 2008).

2. Materials and methods

Sixteen research corneas (6 pairs and 4 single corneas), fromdonors ranging in age from 19 to 81 years old (mean: 57.6 years)were obtained from various eye banks. Seven of the corneas wereoriented at time of harvesting by placing an indelible ink mark atthe superior pole. The remaining nine corneas were not oriented.Most of the corneas (n ¼ 14) were placed directly into roomtemperature 10% neutral buffered formalin; two corneas wereplaced in optisol preservative medium for 6 h prior to immersionfixation. Death-to-preservation (DTP) times ranged from 4.5 to18.0 h. All corneas were shipped overnight in cold fixative solutionto our laboratory at which time they were transferred immediatelyinto ice cold 0.1 M phosphate buffered saline (PBS) containing 30%w/v sucrose.

In preparation for immunohistochemical staining, fourteen ofthe corneas were cut into three large standardized pieces. Thecentral corneal button was removed with a 6.0 or 6.5 mm cornealtrephine. The remaining cornea, comprised of a 2.0e3.0 mm widerim of peripheral cornea and about 0.5e1.0 mm of attached sclera,was then cut with a razor blade into nasal and temporal halves(oriented corneas) or two random halves (non-oriented corneas).Each of the three pieces was sectioned tangentially at �20 �C ina cryostat according to the following protocol. Each specimen wasplaced on a glass microscope slide and pressed, epithelial sidefacing down, on a flat platform of frozen OCT compound ona cryostat chuck. A total of four 100 mm-thick sections were then cutfrom the posterior surface of each central corneal button, and five

or six 100 mm-thick sections were cut from the posterior surface ofeach peripheral corneal rim. All sections were collected in serialorder in ice cold 0.1 M PBS. Tissue sectioning was then halted andthe residual corneal specimen, still embedded in its frozen OCTmatrix on the cryostat chuck, was immersed in a petri dish filledwith ice cold PBS. As the OCT compoundmelted, the residual tissue,consisting of the entire corneal epithelium and approximately50e100 mm of anterior-corneal stroma, was released intact into thePBS. The specimens thus obtained will be referred to henceforth inthis paper as “anterior-cornea whole mounts”.

To increase their permeability, all anterior-corneawholemountsand 100 mm-thick stromal sections were incubated overnight at37 �C in 0.01% hyaluronidase (type IV-S, SigmaeAldrich, Inc., St.Louis, MO) and 0.1% ethylenediaminetetraacetic acid (EDTA; Sigma)in 0.1 M PBS pH 5.3 (Barrett et al., 1999). The next morning thetissues were rinsed three times for 15 min each in PBS containing0.3% Triton X-100 (PBS-TX), and incubated for 2 h in blocking serum(1% bovine serum albumin in PBS-TX). The tissues were thenincubated overnight at room temperature (RT) on a rocker table ina mouse monoclonal antibody directed against neuronal class IIIbeta-tubulin (TuJ1, 1:500, Covance Research Products, Berkeley,CA). After three more PBS-TX rinses, the tissues were incubated for2 h at RT in secondary antibody (biotinylated horse anti-mouse IgG,1:200; Vector Laboratories, Burlingame, CA), rinsed again in PBS-TX, and incubated for 2 h at RT in avidin-biotin-horseradishperoxidase complex (ABC reagent; Vector Laboratories). After threemore PBS-TX rinses, the tissues were incubated for 8 min at RT in0.1% diaminobenzidine (Sigma) and 0.009% H202 and then rinsedthree times in PBS and twice in distilled water. The tissues werethenmounted in serial order on chrome alum-gelatin coated slides,air-dried, dehydrated in graded alcohols, cleared in xylene, andcoverslipped with Permount under weighted coverslips.

Two additional, non-oriented corneas were processed sepa-rately from those described above and were used mainly to studythe distances from the ocular surface at which stromal nervebundles entered the peripheral cornea. Each of these two corneaswas cut from apex to limbus with a razor blade into eight, equal-sized wedge-shaped pieces. Each wedge was then frozen in OCTcompound and six serial sections per specimen were prepared ina cryostat by cutting the tissue parallel to the corneoscleral limbusat a distance precisely 6.0 mm from the corneal apex. The sectionswere collected in PBS and processed free-floating for neurotubulinimmunohistochemistry as described above except without hyal-uronidase/EDTA pretreatment.

2.1. Qualitative observations

Qualitative analyses of corneal nerve architecture andmorphology were performed using a Leica DM4000 researchmicroscope. Comprehensive schematic line drawings of cornealmain stromal bundles, midstromal plexus, subepithelial plexus,subbasal nerve plexus, and intraepithelial terminals were preparedat final magnifications of 30e300� by using a drawing tubeattached to the microscope. Color images were captured witha Leica DFC420C digital camera.

2.2. Quantitative assessments

2.2.1. Main stromal nerve bundlesThe main stromal nerve bundles were analyzed at their point of

entry from the corneoscleral limbus into the peripheral cornea. Fivedistinct parameters were assessed: total number, distribution,spacing between adjacent bundles, diameter, and distance from theocular surface. The first three parameters were determined byplotting the locations of all main stromal nerve bundles in the

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anterior-corneawholemount and serial 100 mm-thick sections ontocomposite illustrationsmadewith the aid of a drawing tube at afinalmagnification of 80�. The location of the corneoscleral limbus wasapproximated on each completed illustration by drawing a 12.0mmdiameter circle centered on the corneal apex. The total number ofstromal nerve bundles, and the distances between adjacent bundlesat their points of intersectwith the edge of the circle, were recorded.The diameters of 130 randomly selected main stromal nerves fromfour different corneas were determined at their points of entry intothe peripheral cornea by using the measuring tool device of Image J(NIH) on calibrated digital images taken on a Leica microscope ata magnification of 40�.

The depths at which the main stromal nerve bundles enteredthe cornea at the limbus were investigated in 30 mm-thick sectionscut parallel to the corneoscleral limbus. The best section from eachcorneal wedge, as judged by nerve staining quality and lack ofsectioning artifacts, was selected for study. The distance from theepithelial surface to the midpoint of each main stromal bundlecontained within that section was measured by using the Image Jmeasuring tool.

2.2.2. Subepithelial plexus (SEP)Nerve fiber diameters in four corneas were determined from

digital images of the SEP taken with a 40� objective. The SEPcontained two main types of nerve fibers (see Results): straight orcurvilinear nerves that penetrated Bowman's membrane to enterthe corneal epithelium, and tortuous anastomotic nerves thatremained confined in their distribution to the SEP. The diameters of100 fibers from each population were determined by using themeasuring tool device of Image J on calibrated images. The diam-eters of the straight or curvilinear nerves were measured within10e50 mm of where the fibers penetrated Bowman's membrane.

2.2.3. Bowman's membrane penetration sitesThe sites where stromal nerve fibers passed through Bowman's

membrane and continued into the epithelium as subbasal nervesare known as Bowman's membrane “penetration sites.” The totalnumber, distribution, and density of penetration sites were deter-mined in the central 10mm areas from three anterior-corneawholemounts by plotting the locations of every penetration site onto highmagnification line drawings prepared with a drawing tube. Sub-basal nerves in the perilimbal cornea, most of which originateddirectly from the limbal plexus, were not included in these counts.

Fig. 1. a. Main stromal bundles (arrows) entering the peripheral cornea at the corneosclerstromal bundle in a 100 mm-thick section.

2.2.4. Subbasal nerve fibersSubbasal nerve fiber density (NFD) and diameters were deter-

mined in the central cornea of six donors aged 19e78 years old(mean age, 51.3 years). For NFD measurements, a 0.5 mm2 rectan-gular area (1.0 mm horizontal� 0.5 mmvertical) centered preciselyon the corneal apex was designated for analysis. Digital images ofthe central subbasal nerve plexus were taken on a light microscopewith a 10� objective and enlarged to a final magnification of 200�.All subbasal nerves and interconnecting fibers located within the0.5 mm2

field were traced carefully with a calibrated tracing tool(Image J). Nerve fiber density was reported as the total combinedlength in millimeters of all subbasal and interconnecting nervefibers within the 0.5 mm2 sample area and reported as mm/mm2.

Subbasal nerve diameters were measured in photomontages ofthe central cornea (4.0 mm horizontal � 0.5 mm vertical) takenwith a 40� objective. A 3 mm horizontal line centered on thecorneal apex was positioned across the montage using AdobePhotoshop. The diameter of every subbasal nerve or interconnect-ing fiber that transected the horizontal guide line was determinedat its point of intersect with the line by using the measuring tool ofImage J. A total of 460 subbasal nerves and 97 interconnectingfibers were measured.

2.2.5. Epithelial nerve terminal densityEpithelial nerve terminal densities in small areas of the central

cornea in three anterior-cornea whole mounts were determined bymapping the location and morphology of all nerve terminals in thesuprabasal cell layers onto high magnification line drawingsprepared with the aid of a drawing tube attached to the lightmicroscope. The areas that were evaluated measured 1.0 mm2 (twocases) or 0.4 mm2 (one case) and were selected on the basis ofoptimal nerve terminal staining quality.

3. Results

3.1. Technical note: effect of death-to-preservation timeon corneal nerve staining

Anterior-cornea whole mounts with death-to-preservation(DTP) times between 7 and 13 h (n ¼ 6) yielded optimal nervestaining and provided superior demonstrations of the corneal SEP,subbasal nerves and intraepithelial terminals. Anterior-cornealwhole mounts with DTP times in excess of 13 h (n ¼ 6) also

al limbus in an anterior-cornea whole mount. b. High magnification image of a main

Fig. 2. Main stromal nerve bundles enter the peripheral cornea uniformly from alldirections. The dashed line indicates the approximate location of the corneosclerallimbus.

Fig. 4. Distances from the corneal surface at which main stromal nerve bundles enterthe peripheral cornea at the corneoscleral limbus. n ¼ 131 nerves.

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contained numerous intensely stained nerves; however, manyepithelial nerves showed varying signs of degeneration, such asswelling of subbasal nerves and fragmentation or loss of intra-epithelial terminals. Anterior-corneawhole mounts with DTP timesless than 7 h (n ¼ 4) were unexpectedly resistant to immunohis-tochemical staining and contained very few well-stained subbasalnerves or intraepithelial terminals, except at cut edges of the tissueor in areas where the epithelium had been damaged inadvertentlyduring processing.

3.2. Main stromal nerve bundles

A mean of 71.3 � 14.3 main stromal nerve bundles (range,53e89) entered the human cornea at the corneoscleral limbus

Fig. 3. Main stromal nerve bundles (arrows) entering the peripheral cornea near thecorneoscleral limbus. In some instances, multiple stromal bundles enter the cornea atthe same location but at different depths (right side of figure).

(Fig. 1). The nerve bundles were distributed uniformly about thecorneal circumference (Fig. 2) and were separated from oneanother by a mean distance of 0.48 mm � 0.40 mm. The stromalbundles entered the peripheral cornea at a mean distance of293 � 106 mm from the corneal surface; however, occasionalbundles entered as far anterior as 56 mm or as deep as 543 mm(Figs. 3 and 4). Themean diameter of the main stromal bundles was20.3 � 7.0 mm (Fig. 5).

3.3. Midstromal nerve plexus

Soon after entering the cornea, each stromal nerve bundle gaverise through repetitive branching to varying numbers of progres-sively smaller and smaller stromal nerves that anastomosedfrequently, often at highly acute branch points, to form a moder-ately dense midstromal plexus. The distal branches of the mid-stromal nerves often coursed centrally for several millimeters andon occasion crossed the geographic center of the cornea to reachthe opposite side. The midstromal plexus in the peripheral stromaoccupied roughly the anterior one-half of the stroma while in thecentral cornea the plexus occupied approximately the anterior one-third. On rare occasions a few isolated nerves were observed in theposterior half of the stroma; however, nerves were never seenadjacent to Descemet's membrane or in the corneal endothelium.

Themidstromal plexus was most dense in the peripheral corneaand decreased progressively in density and anatomical complexity

Fig. 5. Main stromal nerve bundle diameters.

Fig. 6. Low magnification survey image of the peripheral corneal innervation. Mainstromal bundles (msbs) branch and give origin to a moderately dense midstromalplexus composed of straight/curvilinear fibers (arrowheads) and tortuous fibers(arrows). Some of the straight or curvilinear fibers penetrate Bowman's membrane andgive rise in a more superficial plane to subbasal nerve fibers (snf).

Fig. 8. Stromal free nerve endings (arrows).

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in a central direction. In the peripheral cornea, the plexus wascomprised mainly of medium and small diameter nerves withstraight or curvilinear trajectories, and varying numbers of tortuousfibers (Fig. 6). In the central cornea, the midstromal plexus was lessdense and consisted mostly of small diameter, curvilinear nerves(Fig. 7). A few nerve fibers in all areas of the cornea terminated inthe stroma as free nerve endings (Fig. 8).

3.4. Subepithelial plexus

Most midstromal nerve fibers continued into the narrow bandof anterior stroma located immediately beneath Bowman'smembrane and gave rise to a dense, roughly two-dimensional,

Fig. 7. Mid-stromal plexus in the central and pericentral cornea. The area illustrated is6 mm in diameter and is centered on a point 0.5 mm from the corneal apex (asterisk).The drawing is a composite of an anterior-cornea whole mount and two 100 mm-thickstromal sections and illustrates in two-dimensions the entire midstromal nerve plexus.Deeper sections of the corneal stroma from this specimen contained no nerve fibers.The distal continuations of the midstromal nerves (e.g., arrows) give origin to thesubepithelial plexus (not illustrated). The area inside the box is illustrated in greaterdetail in Fig. 12.

subepithelial plexus (SEP). The SEP contained two main types ofnerve fibers: a modest population of straight or curvilinear nerves,and a more abundant and anatomically complex population oftortuous, highly anastomotic nerves (Fig. 9a, b). The straight orcurvilinear anterior stromal nerves ranged in size from 0.37 to9.06 mm in diameter and had a mean diameter of 4.09 � 2.15 mm(Fig.10). Most of these nerves penetrated Bowman'smembrane andcontinued into the corneal epithelium as subbasal nerves (seebelow). Other straight or curvilinear nerves gave origin at theirdistal termini to the tortuous SEP nerve fibers. The tortuous SEPfiber population consisted of mixtures of small and mediumdiameter fibers that ranged in size from 0.24 to 3.28 mm. The nervescoursed in seemingly random directions and often anastomosedextensively to form complex, grid-like meshworks (Fig. 11). Thedensity and anatomical complexity of the SEP tortuous fiber pop-ulation varied considerably from cornea to cornea and from regionto region in the same cornea. In general, the SEP was much moredense in the peripheral and intermediate cornea, and less dense,inconsistent, and patchy in the central cornea (Fig. 12). It wasimpossible because of the plexiform nature of the SEP (e.g., Fig. 11)to determine how most tortuous nerves terminated; however,a few terminated in the subepithelial stroma as free nerve endings,and a very small number (typically only 1e4 per cornea) gave originto collaterals that penetrated Bowman's membrane and formedsubbasal nerves.

3.5. Nerve penetration into the corneal epithelium

Relativelymodest numbers of nerve fibers in the SEP penetratedBowman's membrane to give rise to subbasal nerves (Fig. 13). Amean of 204 � 58.5 SEP nerves (range: 156e269) penetratedBowman's membrane inside the 10 mm diameter zone centered onthe corneal apex; this corresponds to an overall density of2.60 penetrations/mm2 (Fig. 14). The density of nerve penetrationsites was about twice as high (3.18/mm2 vs. 1.55/mm2) in the per-icentral cornea (3e5 mm from the corneal apex) than in the centralcornea (0e3 mm from the apex). Nerve penetration density waslowest in the area surrounding and inferonasal to the corneal apex.Additional subbasal nerves in the extreme peripheral (i.e., peril-imbal) corneal epithelium originated directly from the limbalplexus or from short, radially directed collaterals of limbal nerves(Fig. 15). The latter penetrations were difficult to quantify and arenot included in the data shown in Fig. 14.

3.6. Subbasal nerve plexus

Immediately after penetrating Bowman's membrane, eachstromal nerve branched into one or more subbasal nerves that

Fig. 9. a. Peripheral corneal innervation, focused on the subepithelial plexus. The boxed area near the upper left corner of fig a is shown at higher magnification in b. a. The SEPconsists of modest numbers of straight or curvilinear fibers (arrowheads) and a dense, plexiform network of tortuous nerve fibers (arrows). b. SEP straight or curvilinear fibers(arrowheads) penetrate Bowman's membrane (at open circles) to give rise to subbasal nerves. The tortuous nerve fibers (arrows) anastomose frequently and give the SEP its highlycharacteristic plexiform appearance.

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coursed parallel to the ocular surface near the interface of Bow-man's membrane and the basal epithelium (Fig. 16). Most of thesubbasal nerves observed in this study were of smooth caliber;however, some were beaded in appearance. The term “epithelialleash” is defined as a group of subbasal nerves that derives from thesame parent anterior stromal nerve (Rozsa and Beuerman, 1982;Schimmelpfennig, 1982; Chan-Ling, 1989). At their points of origin,each leash consisted initially of 1e20 subbasal nerves; however, theabsolute number of subbasal fibers in a given leash fluctuatedcontinuously in the proximo-distal direction due to repetitivenerve branching and anastomotic connections via thin, obliquely-oriented interconnecting fibers. Epithelial leashes in the central

Fig. 10. Diameters of SEP straight and curvilinear nerve fibers. Measurements weretaken within 50 mm of where the nerves penetrated Bowman's membrane.

Fig. 11. Schematic line drawing of a small area of the SEP located approximately 3 mmfrom the corneal apex. Abundant, thin tortuous nerve fibers anastomose frequently toform a dense, felt-like meshwork. Embedded within this meshwork are two straight/curvilinear nerves (arrows) that penetrate Bowman's membrane (open circles) andcontinue into the basal epithelial layer as subbasal nerves (e.g., arrowheads).

Fig. 12. High magnification line drawing of the midstromal and subepithelial plexusesin the central corneal region indicated by the box in Fig. 7. SEP density in the centralcornea is often patchy in nature and areas of moderate-to-high tortuous nerve fiberdensity (e.g., large asterisks) often intermingle with areas of lower tortuous nerve fiberdensity (smaller asterisks) in seemingly random fashion.

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and intermediate cornea anastomosed extensively with oneanother in both the lateral (side-to-side) and proximal-to-distalaxes to form a dense, homogenous subbasal nerve plexus in whichclear-cut boundaries between adjoining leashes were no longerdiscernible (Fig. 17). Epithelial leashes in the peripheral cornea, incontrast, were less numerous, more widely separated, demon-strated fewer side-to-side anastomoses, and contained few thicksubbasal nerves (Fig. 18).

When viewed in its entirety, the subbasal nerve plexuscomprised a gentle, spiral-like assemblage of long, curvilinearsubbasal nerve fibers that converged on an imaginary center, orvortex, located inferior and slightly nasal to the corneal apex(Figs. 19 and 20). As a consequence of the whorl-like arrangement,

Fig. 13. Stromal nerve penetrations through Bowman's membrane in anterior-cornea whonerve (arrow) penetrates Bowman's membrane (open circle) and branches into multiplebranches (arrowheads) immediately prior to, or while penetrating, Bowman's membrane. Epenetrates Bowman's membrane (asterisk) and continues as a subbasal nerve (arrowheads

subbasal nerves in the region of the corneal apex coursed ina predominantly near-vertical (1 o'clock-to-7 o'clock) direction,while subbasal nerves in the intermediate and peripheral corneacoursed in various directions consistent with their relative posi-tions within the whorl. Subbasal nerves in the extreme peripheralcornea, especially in the inferonasal quadrant, often coursedparallel to the limbus for varying distances before changing direc-tion gradually or abruptly to reorient towards the vortex (Figs. 15and 21).

The geographical center of the subbasal nerve vortex waslocated between 2.18 and 2.92 mm (mean ¼ 2.51 mm � 0.23 mm)from the corneal apex. Near the center of the vortex, the distalsegments of the subbasal fibers in some corneas fused to form ananastomotic network that spiraled gently in either a clockwise (twocorneas, one OD one OS) or counterclockwise (one cornea, OD)direction (Fig. 22). In other cases (four corneas), the subbasal nervesdid not form a prominent spiral but ended on opposing sides of animaginary seam-like interface (e.g., Fig. 19).

The mean subbasal nerve fiber density in the central corneasfrom six different donors was 45.94 � 5.20 mm/mm2 (range:39.6e53.3 mm/mm2). No correlation between subbasal nervedensity and donor age was observed (Fig. 23). A horizontal linedrawn through the corneal apex intersected a mean of 25.83� 4.34subbasal nerves/millimeter (not including interconnecting axons).Mean subbasal and interconnecting nerve fiber diameters in thecentral corneawere 1.51�0.74 mmand 0.69� 0.26 mm, respectively(Figs. 24 and 25). Subbasal nerve fiber length varied widely. Theshortest subbasal nerves measured less than 1 mm in total lengthand were concentrated near the center of the vortex (e.g., Fig. 22b)and in the perilimbal region. The longest subbasal nerves wereconcentrated in the superior corneal quadrant (e.g., Fig. 19) andoften traveled distances of 6.5e8.0 mm or longer.

3.7. Intraepithelial terminals

Intraepithelial terminals were observed in all corneas examinedin the current study; however, the morphological integrity of thesedelicate structures variedwidely from cornea to cornea in amannerthat did not correlate well with death-to-preservation time. Thedescriptions that follow are based on observations from three

le mounts (aec) and in a 30 mm-thick perpendicular section (d). a. A straight stromalsubbasal nerves (arrowheads). b, c. Some stromal nerves (arrows) split into multipleach branch then gives rise to one or more subbasal nerves. d. A stromal nerve (arrow)).

Fig. 14. a. Location of stromal nerve penetrations (solid circles) through Bowman's membrane in a 48-year-old cornea. The area illustrated is 10 mm in diameter and centered on thecorneal apex (A). V, center of subbasal nerve vortex. The numbers, 1 through 5, indicate distance in millimeters from the apex. b. Density of Bowman's membrane nerve penetrationsites as a function of distance from the corneal apex. n ¼ 611 nerve penetration sites from three different corneas.

C.F. Marfurt et al. / Experimental Eye Research 90 (2010) 478e492 485

corneas in which the majority of intraepithelial terminals werereasonably well preserved. The term “intraepithelial terminal” asused in this study refers to the entire epithelial axon distal to itspoint of origin from a subbasal nerve and includes all of its collat-eral branches and terminal expansions (“nerve endings”).

Intraepithelial terminals originated exclusively as branches ofsubbasal nerves. The terminals were distributed abundantly

Fig. 15. Nerve entry into the peripheral corneal epithelium. Subbasal nerves in theperipheral cornea originate either directly from the limbal plexus (e.g., arrows), orfrom short, radially directed branches of the limbal plexus (arrowheads). Open circles,stromal nerve penetrations through Bowman's membrane. A main stromal bundle(msb) is visible entering the cornea from the limbus in a deeper plane of focus.

throughout all layers of the epithelium and varied considerably intotal length, predominant directional orientation, and morpho-logical complexity. Terminals in the basal epithelial cell layergenerally coursed parallel to the parent subbasal nerve fibers,branched relatively infrequently, and gave rise to small numbers ofbulbous nerve endings (e.g., Fig. 18). Intraepithelial terminals inmore superficial epithelial layers were generally more complex.Some of latter axons ended as single terminal expansions; however,the majority branched one or more times at acute angles andsupported small numbers of variously oriented, preterminalcollaterals in complex treelike morphologies (Figs. 26 and 27). Insome cases, groups of intraepithelial terminals that originated fromthe same subbasal nerve were oriented in a remarkably uniformdirection (Fig. 28).

Nerve terminal density in the wing and squamous cell layers ofthe central cornea was 37.17 mm/mm2, or 605.8 terminals/mm2.The total lengths of intraepithelial terminals in the wing andsquamous cell layers, including all collateral branches and nerveendings, ranged from 9.0 to 780.0 mm.

3.8. Summary of major findings

The major findings of this study are summarized in Table 1.

4. Discussion

The results of this study provide a detailed and comprehensivedescription of the human corneal innervation that adds to existingknowledge in the field. The use of immunohistochemically stained,thick anterior-cornea whole mounts made it possible to visualizethe entire corneal innervation, except for the deepest stromalnerves, in a single preparation and revealed important three-dimensional relationships among midstromal nerves, SEP, subbasalplexus and epithelial nerve terminals. Diaminobenzidine (DAB)wasused to label the nerves in this study because the chromogen ispermanent and does not fade under prolonged illumination; thismade it possible to prepare large-format, detailed illustrations ofthe immunohistochemically stained nerves with a drawing tubeattached to the light microscope.

The results of this study demonstrated a critical and unexpectedrelationship between death-to-preservation (DTP) time andsuccessful corneal nerve staining. Anterior-cornea whole mounts

Fig. 16. Subbasal nerve fibers in an anterior-cornea whole mount (a) and in a perpendicular, 30 mm-thick section (b). a. An anterior stromal nerve (arrow) divides and penetratesBowman's membrane (open circles) to form an “epithelial leash” comprised of approximately 16e18 subbasal nerve fibers. The black line shows the approximate plane of section ofthe 30 mm-thick section (from a different cornea) shown in figure b. b. Subbasal nerve fibers (e.g., arrows) travel roughly parallel to one another in the basal epithelial cell layer closeto Bowman's membrane (bm). A small diameter stromal nerve (arrowhead) penetrates Bowman's membrane near the center of the field. e, corneal epithelium. s, stroma. Calibrationbar equals 20 mm in both figures.

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with DTP times between 7 and 13 h provided the best results,whereas wholemounts with DTP times less than 7 h contained veryfew well-stained epithelial nerves. The latter observation suggeststhat some postmortem degradation of the corneal epithelium priorto immersion fixation may be required for successful hyaluronidasepenetration and permeabilization of the thick specimens.

4.1. Stromal nerve bundles

The mean number of main stromal nerve bundles that supplythe human corneas examined in this study (71.3 � 14.3) is slightlyless than the 80 bundles per cornea reported by Zander andWeddell (1951), and greater than the 44 bundles reported byAl-Aqaba et al. (2009). The main stromal nerve bundles seen here

Fig. 17. a. Detailed schematic line drawing of the central subbasal nerve plexus. The area ilregion shown in Fig. 12. b. Digital image of a small area of the subbasal nerve plexus illustrasubbasal nerve fiber. Arrowheads, interconnecting fibers.

are distributed uniformly around the corneal circumference andenter the cornea radially from all directions (Al-Aqaba et al., 2009).This observation contradicts previous statements that stromalnerves enter the human cornea in greater numbers at the nasal andtemporal poles (Darwish et al., 2007; Muller et al., 2005; Solomonet al., 2004). The reason for this apparent discrepancy is not known.The human cornea receives most of its sensory innervation fromtwo long ciliary nerves that enter the posterior globe medial andlateral to the optic nerve and course forward in the suprachoroidalspace at the nasal and temporal meridians (Bron et al., 1997;Vaughn, 1992). Prior to reaching the corneoscleral limbus, thenerves branch repetitively into smaller bundles and anastomoseextensively with branches of the short ciliary nerves (May, 2004;Trivino et al., 2002) to form 60e80, uniformly distributed nerve

lustrated is 2.28 mm wide � 2.80 mm high (6.38 mm2) and is from the same cornealted schematically in figure a. Large arrow, thick subbasal nerve fiber. Small arrows, thin

Fig. 18. Subbasal nerves in the peripheral cornea. The SNFs anastomose less frequentlyand are generally thinner and more uniform in diameter than are central SNFs. Arrows,nerve terminals in the basal epithelial cell layer (see later text for details).

Fig. 20. Low magnification photomontage of the subbasal nerve plexus in a 6 mmdiameter, anterior-cornea whole mount from a 48-year-old cornea. The orientationnotch at the top of the whole mount is the superior pole.

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bundles that approach the limbus radially from all directions.Clinical observations seem consistent with this view, in that blockexcisions of anterior uveal tumors or epithelial ingrowths locatednear the limbus at the medial or lateral poles are not associatedwith higher incidences of neuroparalytic keratitis (Groh et al.,2002).

The results of the present study may have implications forunderstanding the sensory loss that occurs following cataractsurgery. The mean distance between main stromal bundles repor-ted in the present study is about 0.5 mm; thus, a 2.8e3.0 mm longcurvilinear, clear corneal incision such as is commonly used forfoldable and injectable intraocular lenses would transect a mean ofw6 major stromal nerve bundles, or approximately 6e11% of thetotal corneal innervation. Following phacoemulsification, cornealsensitivity is reduced in the vicinity of the incision and, to a morevariable degree, in the central cornea (Khanal et al., 2008; Sito-mopul et al., 2008). This sensory loss is most likely explained bycombined damage to main stromal bundles and subbasal nerves inthe vicinity of the incision.

Fig. 19. Subbasal nerve plexus in a 6 mm diameter central button from a 61 year-oldcornea. For clarity, only the largest diameter subbasal nerve fibers have been illus-trated. Individual subbasal nerves follow straight or curvilinear trajectories andconverge on an imaginary center, or vortex (asterisk), located approximately 2.5 mminferonasal to the corneal apex.

The results of this study also suggest that transection of mainstromal bundles may produce a significant corneal denervationduring LASIK surgery. The most common corneal flap thicknessused in LASIK surgery is approximately 160 mm and photoablationmay remove as much as an additional 100 mmof stromal tissue. Theresults of our study showed that approximately 37% (48 out of 131)

Fig. 21. Subbasal nerves in the perilimbal region of the inferonasal quadrant. Thenerves travel roughly parallel to the limbus for varying distances before altering theirdirection and orienting towards the center of the vortex.

Fig. 22. Subbasal nerve vortices. Subbasal nerve fibers in some corneas rotate counterclockwise (a) while others rotate clockwise (b) about the center of the vortex. The circularregion illustrated in “a” is 400 mm in diameter, centered on the vortex and superior is towards the top. In b, two thick subbasal nerves penetrate the epithelium (arrows) near thecenter of the vortex and arch conspicuously in a clockwise direction.

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of main stromal bundles entered the peripheral cornea at a depth of250 mm or less from the corneal surface and would likely bedamaged by LASIK surgery.

4.2. Midstromal nerves and the subepithelial plexus

The density of the midstromal nerve plexus is greater in theperipheral cornea than in the central cornea. It seems reasonable tospeculate that the central cornea contains fewer stromal nervefibers because most of the epithelial innervation in this regioncomes from stromal nerves that penetrate Bowman's membrane inmore peripheral locations (Fig. 14).

The morphology of the SEP is exceedingly complex and raisesinteresting questions concerning the role of these fibers in cornealneurophysiology. The results of the present study confirm previousIVCM observations (Visser et al., 2009) that the SEP comprises twohighly distinctive types of nerve fibers; however, only the straightand curvilinear nerves provide significant innervation to thecorneal epithelium. The more numerous tortuous nerve fibers,which give the SEP its distinctive morphological appearance,originate as branches of the straight or curvilinear nerves andsupply less than 5% of the epithelial innervation. It was not possibleto determine the ultimate destination of most of the tortuous nervefibers in this study and consequently their functional significanceremains purely conjectural. Some tortuous fibers may constitutea “reserve” population of SEP nerve fibers that assist in epithelial

Fig. 23. Subbasal nerve fiber densities in the central corneas of six donors aged 19e78years old.

reinnervation after corneal injuries. Others may make “bouton enpassant” contacts on resident cells and possess sensory or trophicfunctions. Electron microscopic and IVCM studies have shown thatsome stromal axons form intimate contacts with keratocytes(Matsuda, 1968; Muller et al., 1996; Visser et al., 2009); however,because keratocytes are present throughout the corneal stroma inextremely high density, it remains to be shown whether thesecontacts are functional or coincidental. The relatively smallnumbers of SEP and midstromal “free nerve endings” observed inthe present study suggest that they are likely of limited physio-logical significance.

No attempt was made to calculate SEP density in this studybecause of the high intra- and inter-cornea variability and patchynature of the plexus. The current study confirms previous reportsbased on IVCM that the SEP is denser in appearance in theperipheral cornea and less dense and highly variable in the centralcornea (Auran et al., 1995; Visser et al., 2009; Oliveira-Soto andEfron, 2001). The patchy nature of the SEP in the central corneawasa consistent and factual observation and was not caused byincomplete immunohistochemical staining.

4.3. Stromal nerve penetrations through Bowman's membrane

The results of this study show that the subbasal nerve plexusoriginates from a modest number (mean ¼ 204) of stromal nervesthat penetrate Bowman's membrane within a 10 mm diametercircle centered on the corneal apex. The mean number of

Fig. 24. Subbasal nerve fiber diameters in the central cornea. Subbasal nerves rangedin size from 0.40 to 5.66 mm. n ¼ 460 fibers from six different donors.

Fig. 25. Interconnecting fiber diameters in the central cornea. Interconnecting fibersranged in size from 0.29 to 1.82 mm. n ¼ 97 fibers from four different corneas.

Fig. 27. High magnification schematic line drawing of intraepithelial nerve terminalsin the wing and squamous cell layers of a 1 mm2 area of central cornea.

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penetrations seen here is slightly less than that reported byprevious workers. Ueda et al. (1989) reported a total of 400 pene-trations per cornea, and Al-Aqaba et al. (2009) reported a total of155e185 penetrations within an 8 mm diameter zone centered onthe corneal apex. The differences in the total number of penetrationsites reported in these three studies are most likely explained byvariations in the corneal area examined, and the criteria used tocount the nerve penetration sites. In the present study, a cluster ofpenetrations that originated from the same stromal nerve (e.g.,Fig. 13b, c) was counted as a single “penetration site.”

The results of the present study have shown that nearly 80% ofall Bowman's membrane penetration sites in the central (10 mmdiameter) corneal button are located in the pericentral regionbetween 3 and 5 mm from the corneal apex. Additional largenumbers of subbasal nerves (not quantified in the current study)enter the perilimbal cornea (5e6 mm from the corneal apex)directly from the limbal plexus. Collectively, this high concentrationof pericentral and peripheral subbasal nerves may provide animportant source of central reinnervation after LASIK surgery,penetrating keratoplasty, and cataract surgery. Circular flaps in

Fig. 26. Intraepithelial nerve terminals as seen in an anterior-corneal whole mount (a) andcircle) exclusively as branches of subbasal nerves (arrowheads, deeper plane of focus). Nerveeach branch is capped by a bulbous terminal expansion (arrows). b. Nerve terminals end b

LASIK surgery and graft diameters in penetrating keratoplastyaverage 8.5e9.5 mm and 7.5e8.0 mm in diameter, respectively, andleave intact a 1e2 mm wide peripheral rim of tissue from whichcentral reinnervation occurs slowly via subbasal nerve elongationacross the wound margin (Calvillo et al., 2004; Erie et al., 2005;Niederer et al., 2007; Patel et al., 2007; Tervo et al., 1985).

Nerve penetration sites in the central cornea of living eyesappear by IVCM as bright, irregular or disc-shaped areas approxi-mately 20e40 mm in diameter (Oliveira-Soto and Efron, 2001; Pateland McGhee, 2005). The relative paucity and widespread distri-bution of these penetration sites in the central cornea makes themdifficult to locate by electron microscopy (Matsuda, 1968; Mulleret al., 1996). As the stromal nerve penetrates Bowman's membrane,new axoplasm and membrane are added to the nerve, thus causingthe subbasal nerves to elongate continuously in a proximal-to-distal direction (Auran et al., 1995; Patel and McGhee, 2008).

in a perpendicular, 30 mm-thick section (b). a. Intraepithelial terminals originate (e.g.,terminals in the suprabasal epithelium often possess multiple collateral branches, andlindly as free nerve endings (arrows) in all layers of the corneal epithelium.

Fig. 28. Epithelial nerve terminals. The two peripheral leashes shown here are eachcomprised of only one or two thick subbasal nerves (arrows). Each subbasal nervegives rise to a large cluster of intraepithelial terminals that radiates asymmetrically atroughly right angles to the parent nerve.

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4.4. Architectural organization of the subbasal nerve plexus

The results of the present study have shown that the humansubbasal nerve plexus, when viewed in its entirety over the wholecornea, forms a gentle spiral-like pattern whose center is locatedabout 2.5 mm inferonasal to the corneal apex. This unique

Table 1Summary of quantitative data.

Corneal nerve type and location

Main stromal nerve bundlesTotal number per corneaDiametersDistance between adjacent bundlesDistance from corneal surface at limbal entry point

Subepithelial plexus (SEP)Diameters of straight/curvilinear fibersDiameters of tortuous fibers

Bowman's membrane penetration sites (central 10 mm of cornea)Total numberDensity, central cornea (<3 mm from corneal apex)Density, peripheral cornea (3e5 mm from corneal apex)

Subbasal nerve plexusDistance from corneal apex to center of vortexSubbasal nerve plexus density, central corneaNumber of subbasal nerve fiber intersects/mm, central corneaSubbasal nerve fiber diameters, central corneaInterconnecting fiber diameters, central cornea

Intraepithelial nerve terminals, central cornea, suprabasal layers onlyNerve terminal density (number of terminals/mm2)Nerve terminal density (mm/mm2)Nerve “ending” density (number of endings/mm2)

architectural feature was not recognized in most previous light andelectron microscopic studies because the orientations of thecorneas were in many cases not known and the authors examinedonly limited numbers of small sample areas from the central orpericentral cornea (Muller et al., 1996; Schimmelpfennig, 1982).Muller et al. (2003) proposed a model of subbasal nerve architec-ture that showed subbasal nerves oriented in a superior-to-inferiordirection over the corneal apex, and in a nasal-to-temporal direc-tion in surrounding areas. Patel and McGhee (2005) subsequentlypublished a 5 mm2 IVCM photomontage of the central subbasalplexus in a living eye that confirmed the roughly vertical orienta-tion of subbasal nerves over the corneal apex, and further showedthat subbasal nerves in the central and pericentral corneaconverged in a whorl-like pattern on a point located approximately1e2 mm inferior to the apex. The architectural map of the subbasalnerve plexus demonstrated in the present study confirms the IVCMfindings of Patel and McGhee (2005) and extends their findings toinclude observations on subbasal nerve orientation in moreperipheral and perilimbal corneal regions.

4.5. Subbasal nerve vortex

The morphology and location of the subbasal nerve vorticesdescribed here confirm and extend previous reports of whorl-likepatterns of subbasal nerves located just inferior to the corneal apexin human (Al-Aqaba et al., 2009; Auran et al., 1995; Patel andMcGhee, 2005, 2008; Ueda et al., 1989) and rodent (Dvorscak andMarfurt, 2008; Leiper et al., 2009; Yu and Rosenblatt, 2007)corneas. The mechanisms that govern the formation and mainte-nance of this spiral-like arrangement remain largely conjectural.According to one hypothesis, basal epithelial cells near the cor-neoscleral limbus migrate centripetally in a whorl-like fashiontowards the corneal apex in response to chemotropic guidance,electromagnetic cues, and population pressures (Collinson et al.,2002, 2004; Dua et al., 1996). Subbasal nerves, occupying narrowintercellular spaces within the migratory epithelial sheet (Mulleret al., 1996), are pulled along by mechanical forces and undergocompensatory horizontal elongation (Auran et al., 1995; Patel andMcGhee, 2008). It has also been postulated that shearing forcesexerted on the corneal surface by the eyelids during spontaneous

Mean Range

71.3 � 14.3 53e8920.3 � 7.0 mm 4.77e40.28 mm0.48 � 0.40 mm 0.03e2.61 mm293 � 106 mm 56e543 mm

4.09 � 2.15 mm 0.37e9.06 mmHighly variable 0.24e3.28 mm

204 � 58.5 156e2691.55 � 0.26/mm2 1.34e1.84/mm2

3.1 � 1.04/mm2 2.29e4.32/mm2

2.51 � 0.23 mm 2.18e2.92 mm45.94 � 5.20 mm/mm2 39.62e53.34 mm/mm2

25.83 � 4.34 18.00e29.671.51 � 0.74 mm 0.40e5.66 mm0.69 � 0.26 mm 0.29e1.82

605.8 � 330.0/mm2 331e972/mm2

36.4 � 1.4 mm/mm2 34.9e37.6 mm/mm2

979.7 � 477.9/mm2 593e1514/mm2

C.F. Marfurt et al. / Experimental Eye Research 90 (2010) 478e492 491

blinking may influence the position of the subbasal nerve vortex(Patel and McGhee, 2005).

Elucidation of the mechanisms that govern formation of sub-basal nerve (and epithelial cell) vortices is compounded by theobservation that the vortices do not rotate in the same direction inall corneas. In the present study, conspicuous subbasal nervevortices were observed in less than half of the corneas and whenpresent the nerves rotated clockwise in some cases and counter-clockwise in others. In two other studies of human corneas, thedirection of vortex rotation was reported as clockwise (Al-Aqabaet al., 2009; Patel and McGhee, 2005). Both clockwise and coun-terclockwise rotations, with a preponderance of clockwise rota-tions in most cases, have been reported for rat and mouse subbasalnerves (Dvorscak and Marfurt, 2008; Leiper et al., 2009), humancorneal epithelial cell cultures exposed to static electromagneticfields (Dua et al., 1996), and clinical patients with hurricanekeratopathy, vortex keratopathy, corneal verticillata and toxic epi-theliopathies (Dua and Gomes, 2000; Dua et al., 1993, 1994, 1996).

4.6. Subbasal nerve fiber density and diameters

The mean central subbasal nerve density calculated in thepresent study (45.94 � 5.20 mm/mm2) is approximately two timesgreater than previous high estimates of subbasal nerve density(21.6 � 5.98 mm/mm2 and 25.9 � 7.0 mm/mm2) based on IVCMobservations (Niederer et al., 2007; Patel and McGhee, 2008). Oneexplanation for the higher density seen here may be that theimmunohistochemical staining method used in this study detectssmall diameter subbasal nerves and interconnecting axons that aretoo thin or faint to be imaged reliably by IVCM. Perhaps for the samereason, the mean diameter of central subbasal nerves seen here islower than that reported in most previous studies (see Patel andMcGhee, 2009, for recent review). Thewide range of subbasal nervefiber diameters observed in the present study (0.40e5.66 mm) isconsistent with electron microscopic observations that each sub-basal nerve fiber contains from 1 to 40 individual axons ranging indiameter from0.2 to 0.5mmeach (Matsuda,1968;Muller et al.,1996;Teng, 1962; Ueda et al., 1989).

Most of the subbasal nerve fibers observed in the present studywere of smooth caliber; however, a few beaded axons were alsoseen. Beaded subbasal nerves and SEP fibers have been describedpreviously in healthy living corneas by IVCM (Auran et al., 1995;Oliveira-Soto and Efron, 2001) and in freshly excised corneasexamined by electron microscopy (Muller et al., 1996), but whethersmooth and beaded nerves constitute functionally distinct fibertypes remains unknown. No special considerationwas attributed tothe beaded nerve fiber population in the present study because itwas not knownwith certainty if the latter fibers were “true” beadedfibers or fibers that developed varicosities secondary to post-mortem degenerative changes.

4.7. Intraepithelial nerve terminals

All intraepithelial nerve terminals observed in the current studyrepresent branches of subbasal nerve fibers. This pattern ofepithelial innervation differs from that observed in rabbit corneas(Rozsa and Beuerman, 1982). The latter species contains two pop-ulations of epithelial terminals: those that constitute branches ofsubbasal nerves, and those that ascend vertically into the epithe-lium from the SEP without entering subbasal nerves. These archi-tectural disparities may indicate true interspecies differences, ormay reflect differences in the methods used to stain, image, andanalyze the corneal epithelial terminals. The present study cannotexclude the possibility that nerve terminals entering the

epithelium directly from the SEP may undergo extremely rapidpostmortem degeneration and thus elude detection.

Estimates of central epithelial nerve terminal density in thepresent study (mean ¼ 608 nerve terminals/mm2) are much lowerthan those reported previously in the literature. Schimmelpfennig(1982) reported one gold-chloride stained nerve terminal per 20-micron grid (400 mm2) or about 2500 terminals/mm2 (Oyster,1999). Guthoff et al. (2005) observed 14 fluorescence-stained nerveendings in an area 8800 mm2, or roughly 16,000 nerve endings/mm2. Nerve terminals densities obtained in the present study werederived from line drawing reconstructions of nerve terminals inrelatively large areas (0.4e1.0 mm2) of the central corneal epithe-lium and, similar to previous studies (Schimmelpfennig, 1982;Guthoff et al., 2005) included only nerve terminals located withinthe wing cell and squamous cell layers. The present study did notattempt to quantify nerve terminals in the basal epithelium (e.g.,Fig. 18); however, the latter terminals in rabbits constitute about30% of the total population (Rozsa and Beuerman, 1982).

5. Conclusion

The current investigation used immunohistochemically stainedanterior-cornea whole mounts and thick stromal sections togenerate a detailed and comprehensive description of the humancorneal innervation. The data reported here provide new insightsinto the architectural organization, interrelationships, and densitiesof corneal main stromal nerve bundles, midstromal plexus, sub-epithelial plexus, Bowman's membrane penetration sites, subbasalnerve plexus, and intraepithelial terminals. The informationgenerated from this study may help ophthalmologists to minimizeand better interpret the sensory and trophic consequences ofcorneal nerve damage associated with cataract, refractive, andother ocular surgeries.

Acknowledgements

The authors thank the Lions Eye Institute for Transplant andResearch (Tampa, FL), the Indiana Lions Eye and Tissue TransplantBank, and the San Diego Eye Bank for providing the donor corneasused in this study. The authors also thank Chris Huppenbauer, ChrisBrown, and Jamie Vijay for assistance in the preparation of some ofthe illustrations.

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